Nature's small molecule universe offers a remarkably rich assortment of structural diversity, physicochemical characteristics, and bioactivity. The study of naturally occurring small molecules can be divided into two categories: 1) metabolomics, or study of the small molecule content of cells and tissues to identify (patho)physiologically important biochemical pathways, and 2) natural products discovery, or the identification of compounds possessing biological activity. Ultimately, the investigation of metabolites and natural products depends upon methods that enable their effective separation and enrichment from complex biological samples. Despite considerable effort, global strategies for the enrichment and characterization of these compounds have remained elusive. In this application, we propose to develop an innovative set of technologies to facilitate the enrichment of small molecules of any physicochemical class. Mentored K99 phase: The postdoctoral phase (Aim 1) of the proposal will be carried out under the mentorship of Prof. Benjamin Cravatt at The Scripps Research Institute. The goal of this phase is to develop a chemoselective tagging and enrichment strategy applicable to metabolomic profiling. This training will provide expertise in mass spectrometry, structure determination, and cell culture techniques. The acquired knowledge will provide the foundation for Aims 2 and 3 and transition to an independent career. Independent ROD Phase: Aim 2 will focus on application of the strategy developed in Aim 1 in combination with activity-based proteomic and genomic technologies to assemble biochemical networks that contribute to bacterial persistence. A persistent strain of the hospital pathogen, Pseudomonas aeruginosa, will be examined through collaboration with Prof. Floyd Romesberg. Aim 3 will expand on the small-molecule tagging strategy to create a technology for the enrichment of bioactive natural products. This method will facilitate detection of natural products that are often inaccessible using current purification methods and enable exploration of nature's small molecule repertoire with unprecedented scope and depth. Relevance: This program has the potential to improve public health through development of technologies that will assist in understanding disease pathogenesis, as well as discovery of therapeutic agents. We will apply these methods to the study of persistence and pathogenicity in Pseudomonas aeruginosa and the identification of novel antimicrobials.